Characterisation of AmpC / ESBL genes in some pathogen gram-negatives isolated from clinical cases of livestock and companion animals

Faruk Pehlivanoglu,Ezgi Sababoglu

doi:10.2478/acve-2021-0036

Abstract

Abstract This study was aimed to search and characterize the AmpC and/or ESBL genes of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from clinical cases of local livestock and companion animals between 2017 and 2019. A total of eight ceftiofur-resistant E. coli (n= 7) and ceftiofur-resistant K. pneumoniae (n= 1) and seven P. aeruginosa were isolated from different cases in local animals. By combination disc method, six E. coli isolates and one K. pneumoniae isolate were found to be ESBL producers. By combination of the disc method and double disc synergy test, no P. aeruginosa isolates were found as ESBL producers. In the agar disc diffusion test (ADDT) performed with cefoxitin and cefoxitin-boronic, only one E. coli was determined as AmpC producer. In ESBL-producing isolates, only the CTX-M class gene was detected by polymerase chain reaction (PCR) and subsequent sequence analysis revealed CTX-M-3 and CTX-M-15 variants. An AmpC positive E. coli isolate was found to carry plasmidic ampC gene in cmy-2 variant from CIT family. It was observed that P. aeruginosa isolates did not carry the plasmidic ampC gene. After the chromosomal ampC gene of one P. aeruginosa was amplified by PCR and sequenced, R79Q and T105A mutations in the chromosomal ampC gene was revealed. This showed that overproduction of the ampC enzyme is involved in the resistance to β-lactams in P. aeruginosa isolates in the study.

Highlights

The β-lactamase enzymes produced by Gram-negative bacteria vary widely, and these variations usually develop due to the change in the active site of the enzyme after point mutations
Enterobacterales isolates in the study were found resistant to ceftiofur in the routine antibiotic susceptibility test (ADDT) performed in the laboratory
The number of reports is increasing on these bacteria from diseased livestock and companion animals

Summary

Introduction

The β-lactamase enzymes produced by Gram-negative bacteria vary widely, and these variations usually develop due to the change in the active site of the enzyme after point mutations. AmpC and extendedspectrum β-lactamases (ESBLs) are the most common ones found in bacteria belong to Enterobacterales and other Gram-negative bacteria [1,2]. AmpC-producing bacteria are resistant to most penicillins, cephalosporins (except cefepime and cefpirome), cephamycins and, variable, monobactams [2,3]. ESBLs are generally susceptible to β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam), but AmpC enzymes are not susceptible. AmpC is produced constituvely at low level and contributes to intrinsic resistance to β-lactams. Overproduction of AmpC following mutations in inducible ampC gene (derepressed mutants) creates increased resistance to cephalosporins (including 3rd and 4th generations of cephalosporins), monobactams and antipseudomonal penicillins (antipseudomonal penicillins and piperacillin) [7]

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